Collection and in vitro storage of blood platelets results in the progressive movement of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from inner to outer leaflet of the platelet plasma membrane. This loss of normal plasma membrane phospholipid (PL) asymmetry with exposure of these aminophospholipids on the platelet surface is implicated in the formation of thrombin, in the activation of complement, and in hepato-splenic sequestration and accelerated clearance of platelets following transfusion. This Project aims to identify the mechanism(s) responsible for this abnormal transbilayer movement of plasma membrane phospholipids in stored platelets and to deduce conditions of storage that will preserve normal sequestration of PS and PE to the inner leaflet. The Specific Aims include: (1) To determine whether the progressive movement of PS to the surface of platelets during storage in autologous plasma reflects membrane reorganization induced through mechanical shear, a decrease in activity of the platelet plasma membrane aminophospholipid translocase, and/or inappropriate activation of the platelet plasma membrane phospholipid scramblase; (2) To determine the relative contributions of altered cytosolic Ca2+, ATP, and pH to the accelerated transbilayer movement of platelet plasma membrane phospholipids; (3) To determine how oxidative changes in either PL scramblase or aminophospholipid translocase during cell storage affect transbilayer distribution of plasma membrane phospholipids; (4) To determine the role of plasma components contained in platelet concentrates to the accelerated movement of aminophospholipids to the platelet surface. Of particular interest is the role of thrombin, plasmin and the C5b-9 components of complement; (5) To identify storage conditions optimized to maintain the normal sequestration of platelet plasma membrane aminophospholipids. Those conditions are considered optimal that maximize the activity of aminophospholipid traslocase and minimize that of the intracellular Ca2+-activated PL scramblase.